Vaccination is one of the most effective and economical methods of preventing disease, and multivalent, orally administered vaccines will have the widest clinical applicability. Salmonella have been extensively studied as living vaccine vectors with the potential to carry multiple heterologous antigens, creating multivalent oral vaccines affording protection against several infectious pathogens simultaneously. This proposal describes a translational research program of laboratory and clinical investigation in oral vaccine development using a new rationally attenuated Salmonella typhi vaccine (Ty800) deleted for the phoP/phoQ virulence regulon of Salmonella. Large, single oral doses of S. typhi Ty800 are well tolerated and highly immunogenic in adult human volunteers, suggesting that this strain may be an ideal Salmonella vaccine vector. The goal of this proposal is to genetically engineer Ty800 to express several clinically relevant antigens, including E. coli colonization factor antigen I, a hybrid hepatitis B nucleocapsid-preS antigen, cholera toxin B subunit, and Helicobacter pylori urease. Heterologous antigens will be expressed from prokaryotic promoters on plasmids maintained in Ty800 by balanced lethal complementation of metabolic genes deleted from the bacterial chromosome and repleted by inclusion of the essential metabolic gene on an antigen-encoding plasmid. S. typhi vaccine strains will be evaluated extensively in vitro and in tissue culture systems. Murine analogs of promising constructs will be created in an S. typhimurium strain with the same phoP/phoQ-deleted allele, for optimization of immunogenicity in mice. Promising S. typhi strains will be administered as investigational vaccines to human volunteers, using a successful enteric vaccine testing protocol developed for evaluation of Ty800. The human cellular and humoral immune responses to both the vector strain and heterologous antigens will be studied. Immunological responses to several strains and antigens will be compared in order to determine whether Ty800 can function as a safe and effective live vector for oral delivery of antigens to the human immune system.